Targeting of Tumor Cells by Cell Surface Urokinase Plasminogen Activator-dependent Anthrax Toxin

Liu, Shihui; Bugge, Thomas H.; Leppla, Stephen H.

doi:10.1074/jbc.m011085200

Cited by 160 publications

(177 citation statements)

References 69 publications

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“…These include proteins containing anthrax toxin fused to sequences cleaved specifically by uPA [123] or an-thracycline non-toxic pro-drugs that can be converted to an active parent drug by uPA [123], plasmin [124], or other serine proteinase such as prostate-specific antigen [125,126].…”

Section: Exploitation Of Proteolytic Activitymentioning

confidence: 99%

Role of plasminogen activator-plasmin system in tumor angiogenesis

Rakic¹,

Maillard²,

Jost³

et al. 2003

Cellular and Molecular Life Sciences (CMLS)

118

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New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications.

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Section: Exploitation Of Proteolytic Activitymentioning

confidence: 99%

Role of plasminogen activator-plasmin system in tumor angiogenesis

Rakic¹,

Maillard²,

Jost³

et al. 2003

Cellular and Molecular Life Sciences (CMLS)

118

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“…and also an uncleavable furin site, the pYS54 constructs described above were digested with PstI and HindIII, and the intervening region was replaced with the corresponding PstI to HindIII fragment isolated from a plasmid encoding PA-U7 (12), in which the 164 RKKR 167 sequence is replaced by PGG. The doubly substituted PA proteins derived from PA I656V and PA I656Q were accordingly named PA-U7 I656V and PA-U7 I656Q, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…These analyses require that the protein ligand be a nontoxic variant of the protein of interest. Nontoxic variants of the PA I656Q, PA I656V, and PA TSR proteins were constructed by replacing their furin cleavage sites, 164 RKKR 167 , with the uncleavable sequence PGG of the previously characterized PA-U7 protein (12). The resulting PA-U7 I656Q, PA-U7 I656V, and PA-U7 TSR were used as nontoxic receptor binding competitors.…”

Section: Pad4 Variants Having Substitutions In Multiplementioning

confidence: 99%

“…LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, we demonstrated that a mixture of two PA variants, one requiring activation by matrix metalloproteases and the other by urokinase plasminogen activator, caused strong anti-tumor responses when administered with LF (13, 16).…”

mentioning

confidence: 99%

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Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

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“…In these studies we used PA-U7, 9 the mutant of PA where the furin cleavage site was deleted. PA-U7 retains ability to bind to receptors but cannot be proteolytically activated.…”

mentioning

confidence: 99%